Bioremediation

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Int. J. Mol. Sci. 2011,12, 633-654; doi:10.3390/ijms12010633

International Journal of

Molecular SciencesISSN 1422-0067

www.mdpi.com/journal/ijmsReview

Environmental Applications of Biosurfactants: Recent Advances

Magdalena Pacwa-Pociniczak1, Grayna A. Paza

2,*, Zofia Piotrowska-Seget

1and

Swaranjit Singh Cameotra3

1 Department of Microbiology, Silesian University, Jagielloska 28 street, 40-032 Katowice, Poland;

E-Mails: [email protected] (M.P.-P.); [email protected] (Z.P.-S.)2 Department of Environmental Microbiology, Institute for Ecology of Industrial Areas,

Kossutha 6 street, 40-844 Katowice, Poland3 Institute of Microbial Technology, Sector 39A, Chandigarh-160036, India;

E-Mail: [email protected]

* Author to whom correspondence should be addressed; E-Mail: [email protected];

Tel.: +48-322546031(246); Fax: +48-322541717.

Received: 30 November 2010; in revised form: 8 December 2010 / Accepted: 10 January 2011 /

Published: 18 January 2011

Abstract: Increasing public awareness of environmental pollution influences the search

and development of technologies that help in clean up of organic and inorganic

contaminants such as hydrocarbons and metals. An alternative and eco-friendly method of

remediation technology of environments contaminated with these pollutants is the use of

biosurfactants and biosurfactant-producing microorganisms. The diversity of biosurfactants

makes them an attractive group of compounds for potential use in a wide variety

of industrial and biotechnological applications. The purpose of this review is

to provide a comprehensive overview of advances in the applications of

biosurfactants and biosurfactant-producing microorganisms in hydrocarbon and metal

remediation technologies.

Keywords:biosurfactants; hydrocarbons; metals; remediation technologies

1. Introduction

Biosurfactants are a structurally diverse group of surface-active substances produced by

microorganisms. All biosurfactants are amphiphiles, they consist of two partsa polar (hydrophilic)

OPEN ACCESS


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moiety and non polar (hydrophobic) group. A hydrophilic group consists of mono-, oligo- or

polysaccharides, peptides or proteins and a hydrophobic moiety usually contains saturated, unsaturated

and hydroxylated fatty acids or fatty alcohols [1]. A characteristic feature of biosurfactants is a

hydrophilic-lipophilic balance (HLB) which specifies the portion of hydrophilic and hydrophobic

constituents in surface-active substances.

Due to their amphiphilic structure, biosurfactants increase the surface area of hydrophobic

water-insoluble substances, increase the water bioavailability of such substances and change the

properties of the bacterial cell surface. Surface activity makes surfactants excellent emulsifiers,

foaming and dispersing agents [2]. In comparison to their chemically synthesized equivalents they have

many advantages. They are environmentally friendly, biodegradable, less toxic and non-hazardous.

They have better foaming properties and higher selectivity. They are active at extreme temperatures,

pH and salinity as well, and can be produced from industrial wastes and from by-products. This last

feature makes cheap production of biosurfactants possible and allows utilizing waste substrates andreducing their polluting effect at the same time [37].

Because of their potential advantages, biosurfactants are widely used in many industries such as

agriculture, food production, chemistry, cosmetics and pharmaceutics. The examples of biosurfactant

applications are listed in many review papers [810]. In this review, special attention is paid to the use

of biosurfactants in different aspects of environmental biotechnology. Many properties of microbial

surface active compounds such as emulsification/de-emulsification, dispersion, foaming, wetting and

coating make them useful in physico-chemical and biological remediation technologies of both organic

and metal contaminants. Biosurfactants increase the bioavailability of hydrocarbon resulting in

enhanced growth and degradation of contaminants by hydrocarbon-degrading bacteria present inpolluted soil. In heavy-metal polluted soils biosurfactants form complexes with metals at the soil

interface, which is followed by desorption of the metal and removal from the soil surface leading to the

increase of metal ions concentration and their bioavailability in the soil solution. The new approach is

the use of heavy metal-resistant bacterial strains capable of producing biosurfactants for increasing the

metal-removing efficiency by phytoremediation.

2. Classification and Properties of Biosurfactants

Unlike chemically synthesized surfactants, which are classified according to their dissociationpattern in water, biosurfactants are categorized by their chemical composition, molecular weight,

physico-chemical properties and mode of action and microbial origin. Based on molecular weight they

are divided into low-molecular-mass biosurfactants including glycolipids, phospholipids and

lipopeptides and into high-molecular-mass biosurfactants/bioemulsifiers containing amphipathic

polysaccharides, proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers.

Low-molecular-mass biosurfactants are efficient in lowering surface and interfacial tensions, whereas

high-molecular-mass biosurfactants are more effective at stabilizing oil-in-water emulsions [11,12].

Examples of biosurfactants and their producers are depicted in Table 1.

The biosurfactants accumulate at the interface between two immiscible fluids or between a fluid anda solid. By reducing surface (liquid-air) and interfacial (liquid-liquid) tension they reduce the repulsive


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forces between two dissimilar phases and allow these two phases to mix and interact more easily

(Figure 1) [10].

Table 1.Classificationof biosurfactants and their use in remediation of heavy metal and

hydrocarbon contaminated sites.

BiosurfactantMicroorganism

Applicationsin EnvironmentalBiotechnology

ReferencesGroup Class

Glycolipids

Rhamnolipids

Pseudomonas

aeruginosa,

Pseudomonas sp.

Enhancement of the degradation and

dispersion of different classes of

hydrocarbons; emulsification of

hydrocarbons and vegetable oils;

removal of metals from soil

[1316]

Trehalolipids

Mycobacterium

tuberculosis,

Rhodococcus

erythropolis,

Arthrobacter sp.,

Nocardia sp.,

Corynebacterium sp.

Enhancement of the bioavailability of

hydrocarbons[17]

Sophorolipids

Torulopsis bombicola,

Torulopsis

petrophilum,

Torulopsis apicola

Recovery of hydrocarbons from dregs

and muds; removal of heavy metals

from sediments; enhancement of oil

recovery

[14,18,19]

Fatty acids,

phospholipids

and neutral

lipids

Corynomycolic acid Corynebacterium lepus Enhancement of bitumen recovery [20]

Spiculisporic acidPenicillium

spiculisporum

Removal of metal ions from aqueoussolution; dispersion action for

hydrophilic pigments; preparation of

new emulsion-type organogels,

superfine microcapsules (vesicles or

liposomes), heavy metal sequestrants

[2123]

Phosphati-

dylethanolamine

Acinetobacter sp.,

Rhodococcus

erythropolis

Increasing the tolerance of bacteria to

heavy metals[24]

LipopeptidesSurfactin Bacillus subtilis

Enhancement of the biodegradation

of hydrocarbons and chlorinated

pesticides; removal of heavy metals

from a contaminated soil, sediment

and water; increasing the

effectiveness of phytoextraction

[2527]

Lichenysin Bacillus licheniformis enhancement of oil recovery [28]

Polymeric

biosurfactants

EmulsanAcinetobacter

calcoaceticus RAG-1 Stabilization of the hydrocarbon-in-

water emulsions

[29]

AlasanAcinetobacter

radioresistens KA-53[30]

BiodispersanAcinetobacter

calcoaceticus A2

Dispersion of limestone in water [31]

Liposan Candida lipolyticaStabilization of hydrocarbon-in-water

emulsions

[32]

MannoproteinSaccharomyces

cerevisiae[33]


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Figure 1.Accumulation of biosurfactants at the interface between liquid and air.

The most active biosurfactants can lower the surface tension of water from 72 to 30 mNm1and the

interfacial tension between water and n-hexadecane from 40 to 1 mNm1

[2,10]. Biosurfactantactivities depend on the concentration of the surface-active compounds until the critical micelle

concentration (CMC) is obtained. At concentrations above the CMC, biosurfactant molecules associate

to form micelles, bilayers and vesicles (Figure 2). Micelle formation enables biosurfactants to reduce

the surface and interfacial tension and increase the solubility and bioavailability of hydrophobic

organic compounds [14]. The CMC is commonly used to measure the efficiency of surfactant. Efficient

biosurfactants have a low CMC, which means that less biosurfactant is required to decrease the surface

tension [2]. Micelle formation has a significant role in microemulsion formation [34]. Microemulsions

are clear and stable liquid mixtures of water and oil domains separated by monolayer or aggregates of

biosurfactants. Microemulsions are formed when one liquid phase is dispersed as droplets in another

liquid phase, for example oil dispersed in water (direct microemulsion) or water dispersed in oil

(reversed microemulsion) [2].

Figure 2. The relationship between biosurfactant concentration, surface tension and

formation of micelles [14].

The biosurfactant effectiveness is determined by measuring its ability to change surface and

interfacial tensions, stabilization of emulsions and by studying its hydrophilic-lipophilic


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balance (HLB). The HLB value is a measure to indicate whether a biosurfactant is related to

water-in-oil or oil-in-water emulsion. This factor can be used to determine the suitable applicability of

biosurfactants. Emulsifiers with low HLB are lipophilic and stabilize water-in-oil emulsification,

whereas emulsifiers with high HLB have the opposite effect and confer better water solubility [2,35].

Biosurfactants also influence the bacterial cell surface hydrophobicity (CSH). This ability has been

reported by Al-Tahhan et al. [36], who studied chemical and structural modifications in the CSH of

Pseudomonas aeruginosa by a rhamnolipid in the presence of hexadecane. Results of their study

demonstrated that rhamnolipid, at very low concentration, caused release of lipopolysaccharide (LPS)

from the outer membrane resulting in an increase of cell surface hydrophobicity. In contrast,

Sotirova et al. [37] reported that rhamnolipid at the concentrations below CMC did not affect the LPS

component of the bacterial outer membrane but instead changed the composition of outer membrane

proteins (OMP). However, all of the changes in the structure of the bacterial cell surface cause increase

of accessibility of hydrocarbons to microbial cells.

3. Biosurfactants and Hydrocarbons Degradation/Remediation

The extensive production and use of hydrocarbons has resulted in widespread environmental

contamination by these chemicals. Due to their toxicity, persistent and negative influence on living

organisms it is important to clean-up the polluted sites. Hydrocarbons, as the hydrophobic organic

chemicals, exhibit limited solubility in groundwater and tend to partition to the soil matrix. This

partitioning can account for as much as 9095% or more of the total contaminant mass. As a

consequence, the hydrocarbon contaminants exhibit moderate to poor recovery by physico-chemical

treatments; limited bioavailability to microorganisms; and limited availability to oxidative and

reductive chemicals when applied to in-situand/or ex-situapplications.

3.1. Role of Biosurfactants in Biodegradation Processes

A promising method that can improve bioremediation effectiveness of hydrocarbon contaminated

environments is the use of biosurfactants. They can enhance hydrocarbon bioremediation by two

mechanisms. The first includes the increase of substrate bioavailability for microorganisms, while the

other involves interaction with the cell surface which increases the hydrophobicity of the surface allowing

hydrophobic substrates to associate more easily with bacterial cells [38]. By reducing surface and

interfacial tensions, biosurfactants increase the surface areas of insoluble compounds leading to increased

mobility and bioavailability of hydrocarbons. In consequence, biosurfactants enhance biodegradation and

removal of hydrocarbons. Addition of biosurfactants can be expected to enhance hydrocarbon

biodegradation by mobilization, solubilization or emulsification (Figure 3) [34,3943].

The mobilization mechanism occurs at concentrations below the biosurfactant CMC. At such

concentrations, biosurfactants reduce the surface and interfacial tension between air/water and

soil/water systems. Due to the reduction of the interfacial force, contact of biosurfactants with soil/oil

system increases the contact angle and reduces the capillary force holding oil and soil together. In turn,

above the biosurfactant CMC the solubilization process takes place. At these concentrations

biosurfactant molecules associate to form micelles, which dramatically increase the solubility of oil.

The hydrophobic ends of biosurfactant molecules connect together inside the micelle while the


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hydrophilic ends are exposed to the aqueous phase on the exterior. Consequently, the interior of a

micelle creates an environment compatible for hydrophobic organic molecules. The process of

incorporation of these molecules into a micelle is known as solubilization [42].

Figure 3. Mechanisms of hydrocarbon removal by biosurfactants depending on their

molecular mass and concentration [11,42].

Emulsification is a process that forms a liquid, known as an emulsion, containing very small

droplets of fat or oil suspended in a fluid, usually water. The high molecular weight biosurfactants are

efficient emulsifying agents. They are often applied as an additive to stimulate bioremediation and

removal of oil substances from environments.

In the current literature, the latest advantages of the role of biosurfactants in interaction between

hydrocarbons and microorganisms are presented. Franzetti et al. [17] describe proposed roles for

biosurfactants with respect to their interactions between microorganisms and hydrocarbons in the

content of modulation of cell surface hydrophobicity. High cell-hydrophobicity allows microorganisms

to directly contact oil drops and solid hydrocarbons while low cell hydrophobicity permits their

adhesion to micelles or emulsified oils [17]. They discuss three mechanisms of interaction between

microorganisms and hydrocarbons: access to water-solubilized hydrocarbons, direct contact of cells

with large oil drops and contact with pseudosolubilized or emulsified oil. The authors suggest that

during the different growth stages of microorganisms, biosurfactants can change hydrocarbon

accession modes. In their studies, they observed that Gordonia sp. strain BS 29 grown on hydrocarbons

produced cell-bound glycolipid biosurfactant and extracellular bioemulsifier, and during the phase of

the growth on hexadecane the surface hydrophobicity changes were observed [17,44].

The recent report by Cameotra and Singh [45] throws more light on the uptake mechanism of

n-alkane byPseudomonas aeruginosaand the role of rhamnolipids in the process. The authors reported

a new and exciting research for hydrocarbon uptake involving internalization of hydrocarbon inside the

cell for subsequent degradation. Biosurfactant action dispersed hexadecane into microdroplets,

increasing the availability of the hydrocarbon to the bacterial cells. The electron microscopic studies

indicated that uptake of the biosurfactant-coated hydrocarbon droplets occurred. Interestingly,

internalization of biosurfactant layered hydrocarbon droplets was taking place by a mechanism

similar in appearance to active pinocytosis. This mechanism was not earlier visually reported inbacterial modes for hydrocarbon uptake. Although much work has been done by many groups to


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explain the role of biosurfactants in the degradation of water immiscible substrates, most processes still

remain unclear.

3.2. Biodegradation Studies

The capability of biosurfactants and biosurfactant-producing bacterial strains to enhance organic

contaminants availability and biodegradation rates was reported by many authors [39,41,46].

Obayori et al. [47] investigated the biodegradative properties of biosurfactant produced by

Pseudomonas sp. LP1 strain on crude oil and diesel. The results obtained confirmed the ability of strain

LP1 to metabolize the hydrocarbon components of crude and diesel oil. They reported 92.34%

degradation ofcrude oil and 95.29% removal of diesel oil. Biodegradative properties of biosurfactant

producingBrevibacterium sp. PDM-3 strain were tested by Reddy et al. [48]. They reported that this

strain could degrade 93.92% of the phenanthrene and also hadability to degrade other polyaromatic

hydrocarbons such as anthracene and fluorene.

Kang et al. [49] used sophorolipid in studies on biodegradation of aliphatic and aromatic

hydrocarbons and Iranian light, crude oil under laboratory conditions. Addition of this biosurfactant to

soil increased also biodegradation of tested hydrocarbons with the rate of degradation ranging from

85% to 97% of the total amount of hydrocarbons. Their results indicated that sophorolipid may have

potential for facilitating the bioremediation of sites contaminated with hydrocarbons having limited

water solubility and increasing the bioavailability of microbial consortia for biodegradation.

The effective microbiological method in bioremediation of hydrocarbon polluted sites is the use of

biosurfactant producing bacteria without necessarily characterizing the chemical structure of the

surface active compounds. The cell free culture broth containing the biosurfactants can be applied

directly or by diluting it appropriately to the contaminated site. The other benefit of this approach is

that the biosurfactants are very stable and effective in the culture medium that was used for

their synthesis.

The usefulness of biosurfactant producing strains in bioremediation of sites highly contaminated

with crude petroleum-oil hydrocarbons was confirmed by Das and Mukherjee [50]. The ability of three

biosurfactant producing strains: Bacillus subtilis DM-04, Pseudomonas aeruginosa M and

Pseudomonas aeruginosa NM to remediate petroleum crude-oil contaminated soil samples was

investigated by treating the soil samples with aqueous solutions of biosurfactants obtained from therespective bacteria strains. Additionally, the tested soil was inoculated with mineral-salts media

containing a specified amount of Bacillus subtilis DM-04 or Pseudomonas aeruginosa M and NM

strains. To determine the extent of biodegradation, the soil-phase total petroleum hydrocarbons (TPH)

concentrations were analyzed after 120 days and compared to a control where the soil was treated with

un-inoculated medium. Bioagumentation of studied soil withP. aeruginosa M and NM consortium and

B. subtilis strain showed that TPH levels were reduced from 84 to 21 and 39 gkg1 of soil,

respectively. In contrast, the TPH level was decreased to 83 gkg1in control soil.

Joseph and Joseph [51] separated the oil from the petroleum sludge by induced biosurfactant

production by bacteria. Petroleum sludge is generated in significant amount in the refineries duringcrude oil processing. Crude oil is usually stored in storage tanks. Pollutants present in the oil are

deposited at bottom of the tank. During cleaning of the tank the sludge is recovered and is treated as a


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waste. The sludge used for the investigation contained TPH in the concentration range of

850 150 gkg1. In this study the sludge was inoculated directly with Bacillus sp. strains and by

addition of the cell free supernatant. Un-inoculated sludge was also taken as a control. Upon

inoculation of the supernatant to the sludge slurry, oil separation and reduction of TPH was observed.

The oil separation process was slow initially in the test supplied with the fresh inoculation of the

bacterium compared to the samples inoculated with the supernatant, but the residual TPH of both

became equal within 48 h. The efficiency of removal of the various isolates ranged from 91.67% to

97.46%. Therefore, it has been observed that the biosurfactant produced by the primary inoculum

remained in the supernatant and it was enough to continue the reaction. The biosurfactant displayed the

property to reduce surface and interfacial tensions in both aqueous and hydrocarbon mixtures and

hence had potential for oil recovery.

Biosurfactants have often been used to enhance bioavailability and biodegradation of hydrophobic

compounds but there is little knowledge available about the effect of simultaneous emulsifierproduction on biodegradation of complex hydrocarbon mixtures. Nievas et al. [43] studied the

biodegradation of a bilge waste which is a fuel oil-type complex residue produced in normal ship

operations. Bilge waste is a hazardous waste composed of a mixture of sea-water and hydrocarbon

residue, where n-alkanes, resolvent total hydrocarbons and unsolvent complex mixture are the main

constituents. Unsolvent complex mixtureprincipally is composed by branched and cyclic aliphatic

hydrocarbons and aromatic hydrocarbons, which usually show the greatest resistance to

biodegradation. In their studies, they investigated the biodegradation of an oily bilge wastes by an

emulsifier-producing microbial consortium. As the result for both levels of oily wastes, 136 gkg1of

resolvent hydrocarbons and 406 gkg1of unsolvent mixture, they found that all of the hydrocarbontypes showed an important concentration reduction from their initial values. They observed that the

extent of biodegradation followed the order n-alkanes > resolved total hydrocarbon > unsolvent

complex mixture. An emulsifier-producing microbial consortium used for biodegradation of bilge

wastes showed reduction of n-alkanes, resolvent hydrocarbonsand unsolvent mixture around by 85%,

75% and 58%, respectively.

Barkay et al. [52] tested the effect of a bioemulsifier alasan produced by Acinetobacter

radioresistens KA53 on the solubilization of polyaromatic hydrocarbons (PAHs), phenanthrene (PHE)

and fluoranthene (FLA). They also studied the influence of alasan on mineralization of PHE and FLA

by Sphingomonas paucimobilis EPA505. They indicated that aqueous solubility of phenanthrene and

fluoranthene increased linearly in the presence of increasing concentrations of bioemulsifier (50 to

500 gmL1) and mineralization of PAHs by S.paucimobilis EPA505 was stimulated by appearance

of alasan. The presence of alasan at concentrations of up to 300 gmL1 more than doubled the

degradation rate of fluoranthene and significantly increased the degradation rate of phenanthrene.

Increasing the alasan concentration over 300 gmL1 had no further stimulation on PAHs

mineralization, although solubilization curves showed that the apparent solubility of these compounds

continued to increase linearly with alasan additions in this concentration range. This could be

explained by association of PAHs with multimolecular structures of alasan, formed at concentrations

above the CMC (about 200 gmL1), which was not readily available for the degrading strain.


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Martnez-Checa et al. [53] investigated the usefulness of the V2-7 bioemulsifier producing strain

F2-7 ofHalomonas eurihalinain oil bioremediation process. First, they studied capacity of strain F2-7

to grow and produce bioemulsifier in the presence of different hydrocarbon compounds. They observed

that all analyzed hydrocarbons supported the growth of F2-7 strain and the production of

V2-7 bioemulsifier. The ability of the analyzed strain to remove polycyclic aromatic hydrocarbonswas

investigated during the growth of this strain for 96 h in liquid medium supplemented with naphthalene,

phenanthrene, fluoranthene and pyrene. After the experiment, the obtained residual concentrations of

fluoranthene (56.6%) and pyrene (44.5%) were higher than naphthalene (13.6%) and phenanthrene

(15.6%). Efficiency of strain F2-7 in removing PAHs confirmed its potential applicability in oil

bioremediation technology.

Exopolysaccharide (EPS) secreted by Enterobacter cloacae strain TU was also reported as an

emulsifier [54]. EPS was investigated and was found to have a high emulsifying activity (E24= 75).

The EPS could increase the hydrophobicity of the bacterial cell surface and also neutralize the surfacecharge of the cells.

3.3. Soil Washing Technology

Soil washing technology is characterized by chemico-physical properties of the biosurfactant and

not by their effect on metabolic activities or changes in cell-surface properties of bacteria [55].

However, the processes may enhance the bioavailability for bioremediation. Aqueous solutions of

biosurfactants can be also used to release compounds characterized by low solubility from soil and

other media in process called washing.

Urum et al. [56] investigated the efficiency of different surfactant solutions in removing crude oil

from contaminated soil using a soil washing process. They demonstrated higher crude oil elimination

by synthetic surfactant-sodium dodecyl sulfate (SDS) and rhamnolipid biosurfactants (46% and 44%,

respectively) than natural surfactantssaponins (27%).

Kang et al. [49] analyzed application of sophorolipid, Tween 80/60/20 and Span 20/80/85 as

possible soil washing agents to release 2-methylnapthalene from artificially polluted soil. They

observed that sophorolipid had a higher soil washing efficiency that any other tested nonionic

surfactants except Tween 80. This could be caused by high hydrophilic-lipophilic balance (HLB) of

Tween 80. It appeared that surfactants with a higher HLB resulted in better solubility of2-methylnapthalene.

Lai et al. [57] studied the ability of removing total petroleum hydrocarbon (TPH) from soil by two

biosurfactants: rhamnolipid and surfactin, and two synthetic surfactants: Tween 80 and Triton X-100.

The TPH removal efficiency was examined for low TPH-contaminated (LTC) and high

TPH-contaminated (HTC) soils (containing 3000 and 9000 mgkg1dry soil of TPH, respectively) by

washing them with (bio) surfactant solutions. As a result, they observed that addition of 0.2 mass% of

rhamnolipid, surfactin, Triton X-100 and Tween 80 to LTC soil resulted in a TPH removal of 23%,

14%, 6% and 4%, respectively, while for HTC soil a significantly higher TPH removal efficiency of

63%, 62%, 40% and 35%, respectively, was observed. These results indicated that among four (bio)surfactants, rhamnolipid and surfactin showed superior performance on TPH removal, compared to


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synthetic surfactants. The two biosurfactants examined in this work have the potential to be used as

biostimulation agents for bioremediation of oil-polluted soils.

Franzetti et al. [44] evaluated the application of surface active compounds produced by

Gordonia sp. strain BS29 in soil remediation technologies: bioremediation of soils contaminated by

aliphatic and aromatic hydrocarbons (microcosm bioremediation experiment), and washing of soils

contaminated by crude oil, PAHs, and heavy metals (batch experiment). The work represents the first

study on the potential applications of surface-active compounds produced by Gordonia sp. in

environmental remediation techniques for contaminated soils. In the previous work, surface-active

compounds produced by Gordonia sp. and their role in the access to hydrocarbons were

characterized [58]. The bacterial strain grew on aliphatic hydrocarbons and produced two different

types of surface active compounds: extracellular bioemulsan and cell-bound biosurfactant.

Bioremediation results showed that the bioemulsans produced by Gordonia sp. strain BS29 were able

to slightly enhance the biodegradation of recalcitrant branched hydrocarbons. On the other hand, theauthors obtained the best results in soil washing of hydrocarbons. The mean of the crude oil removal

for bioemulsans was 33%. The study presented by Franzetti et al. [58] showed that the BS29

bioemulsans from Gordonia sp. are promising washing agents for remediation of

hydrocarbon-contaminated soils. The BS29 bioemulsans were also able to remove metals (Cu, Cd, Pb,

Zn, Ni), but their potential in the process was lower than rhamnolipids.

3.4. Clean-up Combined Technology

The aim of the research work reported by Kildisas et al. [59] and Baskys et al. [60] was to develop

inexpensive and efficient combined (complex) technology for cleaning up the soil contaminated by oil

pollutants in a large scale. The described technology was based on bioremediation or phytoremediation

principles and used physical-chemical treatment by washing the contaminated soil. The complex

technology consisted of two stages: at the first stage, the migrating fraction of pollutants was separated

from soil using biosurfactants; at the second stage, the remaining not migrating fraction was rendered

harmless using biodegradation. Phytoremediation was also applied to enhance soil quality. The completed

clean up complex technology is presented by Kildisas et al. [59]. The presented technology consisted of

washing of the migration fraction by application of biosurfactants, separation of water, oil and soil,

biodegradation of residual non-migrating oil fraction by use of specific bacteria with potential to degradethe crude oil and oil products, and phytoremediation. The pilot plant for washing the contaminated soil

was designed and constructed in a space of 340 m2in which 1000 m3of contaminated soil was cleaned

up. In the beginning of the pilot experiment the concentrations of the oil pollutants were between 180

270 gkg1 of soil, and after washing the concentrations were reduced to 3459 gkg1 of soil. After

degradation, the pollutant concentrations dropped to 3.27.3 gkg1of soil [60].

3.5. Microbial Enhanced Oil Recovery (MEOR)

3.5.1. Mechanism of MEOR

Biosurfactants can also be involved in microbial enhanced oil recovery (MEOR). MEOR methods

are used to recover oil remaining in reservoirs after primary (mechanical) and secondary (physical)


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recovery procedures [61,62]. It is an important tertiary process where microorganisms or their

metabolites, including biosurfactants, biopolymers, biomass, acids, solvents, gases and also enzymes,

are used to increase recovery of oil from depleted reservoirs. Application of biosurfactants in enhanced

oil recovery is one of the most promising advanced methods to recover a significant proportion of the

residual oil. The remaining oil is often located in regions of the reservoir that are difficult to access and

the oil is trapped in the pores by capillary pressure [62]. Biosurfactants reduce interfacial tension

between oil/water and oil/rock. This reduces the capillary forces preventing oil from moving through

rock pores (Figure 4). Biosurfactants can also bind tightly to the oil-water interface and form emulsion.

This stabilizes the desorbed oil in water and allows removal of oil along with the injection water [63].

Figure 4.Mechanism of enhanced oil recovery by biosurfactants.

3.5.2. Applications of MEOR

Bordoloi and Konwar [64] investigated the recovery of crude oil from a saturated column under

laboratory conditions. Laboratory studies on MEOR typically utilize core substrates and columns

containing the desired substrate, usually sand. This substrate is used to demonstrate the usefulness of

biosurfactants in recovery of oil from reservoirs. For this purpose, a glass column is packed with dry

sand, then the column is saturated with crude oil and aqueous solution of biosurfactant is poured in the

column. The potential of biosurfactants in MEOR is estimated by measuring the amount of oil released

from the column after pouring the aqueous solution of biosurfactant in the column. The experimentwas carried out in room temperature, 70 and 90 C to evaluate the influence of temperature on

biosurfactant-induced oil recovery. Biosurfactants used in the experiment were produced by bacterial

isolates of P. aeruginosa strains (MTCC7815, MTCC7814, MTCC7812 and MTCC8165).

Biosurfactants of MTCC7815, MTCC7812 and MTCC8165 strains recovered about 4954% of crude

oil from the sand packed column at room temperature; 5257% at 70 C and 5862% at 90 C. The

biosurfactant produced by MTCC7814 was reported to be less efficient. In control samples treated with

culture medium, very little recovery of crude oil was obtained.

Jinfeng et al. [65] evaluated the technical feasibility and effectiveness of improving oil recovery by

microbial enhanced water-flooding techniques in high temperature petroleum reservoirs. The studieswere conducted in Guan 69 Unit in Dagang Oilfield in China by injection of a mixture of

Arthrobacter sp. (A02), Pseudomonas sp. (P15) and Bacillus sp. (B24) strain suspension and the


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nutrient solution through injection wells in an ongoing waterflood reservoir where the temperature

reached 73 C. The pattern of injection nutrient-suspension-nutrient was designed based on the

knowledge of the reservoir conditions and the mechanism of enhancement of oil recovery by the

selected strains in the reservoir. The oil production performance in the unit was periodically monitored

before, during and after microbial water-flooding and then compared. Jinfeng et al. [65] observed that

the oil production steadily increased after microbial water-flooding. The oil production in the unit

before and in the beginning phase of the injection decreased from 55 t/day in January 2000 to 30 t /day

in September 2001, which implies a decline rate of 21%. This situation changed markedly six month

later and by the end of the July 2004, about 8700 t of additional oil was obtained compared with the

predicted oil production. All the seven production wells showed a positive response to the treatment, of

which five wells evidently increased in oil production.

Pornsunthorntawee et al. [66] compared the oil recovery activities of the biosurfactants produced by

Bacillus subtilis PT2 and Pseudomonas aeruginosa SP4 with three synthetic surfactants:polyoxyethylene sorbitan monooleate (Tween 80), sodium dodecyl benzene sulfonate (SDBS) and

sodium alkyl polypropylene oxidebsulfate (Alfoterra). For this purpose, sand-packed column

inoculated with a motor oil complex was used. The surfactant solutions were poured onto the packed

column to test their ability to enhanced oil recovery. The results showed that the biosurfactants

produced by Bacillus subtilis PT2 and Pseudomonas aeruginosa SP4 were more efficient in oil

recovery, removing about 62% and 57%, respectively, of the tested oil. The biosurfactants produced by

Bacillus subtilis PT2 could recover oil more effectively than that produced by

Pseudomonas aeruginosa SP4. In the case of tested synthetic surfactants, the oil recovery was found to

be approximately 5355%.Biosurfactants can also be used to extract hydrocarbon compounds from oil shales in order to utilize

it as a substitute for petroleum energy fuel. In studies conducted by Haddadin et al. [67], biosurfactants

produced byRhodococcus erythropolis andRhodococcus ruber were successfully used for desorption

of the hydrocarbons from El-Lajjun oil shale.

4. Biosurfactants and Metals Remediation

Contamination of soil environments with heavy metals is very hazardous for human and other living

organisms in the ecosystem. Due to their extremely toxic nature, presence of even low concentrationsof heavy metals in the soils has been found to have serious consequences. Nowadays, there are many

techniques used to clean up soils contaminated with heavy metals. Remediation of these soils includes

non biological methods such as excavation, and disposal of contaminated soil to landfill sites or

biological techniques [68]. Biological methods are processes that use plants (phytoremedation) or

microorganisms (bioremediation) to remove metals from soil. Application of microorganisms was

discovered many years ago to help in reduction of metal contamination. Heavy metals are not

biodegradable; they can only be transferred from one chemical state to another, which changes their

mobility and toxicity. Microorganisms can influence metals in several ways. Some forms of metals can

be transformed either by redox processes or by alkylation. Metals can also be accumulated bymicroorganisms by metabolism-independent (passive) or by intracellular, metabolism-dependent


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Int. J. Mol. Sci.2011, 12 645

(active) uptake. Microorganisms can influence metal mobility indirectly by affecting pH or by

producing or releasing substances which change mobility of the metals [69,70].

Two following methods, soil washing or soil flushing, are involved in remediation of metal

contaminated soil. The first technique used is ex situcontaminated soil is excavated, put into the

glass column and washed with biosurfactant solution. In turn, soil flushing of in situ technologies

involves use of drain pipes and trenches for introducing and collecting biosurfactant solution to and

from the soil [15,71]. Interestingly, biosurfactants can be used for metal removal from the soil.

Biosurfactants can be applied to a small part of contaminated soil in which soil is put in a huge cement

mixer, biosurfactant-metal complex is flushed out, soil deposited back, and biosurfactant-metal

complex treated to precipitate out biosurfactant, leaving behind the metal. The bond formed between

the positively charged metal and the negatively charged surfactant is so strong that flushing water

through soil removes the surfactant metal complex from the soil matrix. This method can also be

carried out for deeper subsurface contamination only with more pumping activities.

4.1. Removal of Metals by BiosurfactantsMechanism of the Process

Using biosurfactants have unquestionable advantages because bacterial strains able to produce

surface active compounds do not need to have survival ability in heavy metal-contaminated soil.

However, using biosurfactants alone requires continuous addition of new portions of these compounds.

The usefulness of biosurfactants for bioremediation of heavy metal contaminated soil is mainly

based on their ability to form complexes with metals. The anionic biosurfactants create complexes with

metals in a nonionic form by ionic bonds. These bonds are stronger than the metals bonds with the soil

and metal-biosurfactant complexes are desorbed from the soil matrix to the soil solution due to the

lowering of the interfacial tension. The cationic biosurfactants can replace the same charged metal ions

by competition for some but not all negatively charged surfaces (ion exchange). Metal ions can be

removed from soil surfaces also by the biosurfactant micelles. The polar head groups of micelles can

bind metals which mobilize the metals in water (Figure 5) [38,7173].

Figure 5.Mechanism of biosurfactant activity in metal-contaminated soil [74].


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Int. J. Mol. Sci.2011, 12 646

4.1.2. Applications of the Process

Biosurfactants which are used in bioremediation of metal-contaminated soils have been proposed

for use in metal removal in recent years [72,73]. High potential of biosurfactants in mobilization and

decontamination of heavy metal contaminated soil was confirmed by Juwarkar et al. [75], who used

di-rhamnolipid biosurfactant produced by Pseudomonas aeruginosa BS2 for mobilization of metals

from multi-metal contaminated soil. To study the feasibility of di-rhamnolipid to remove chromium,

lead, cadmium and copper from soil, a column study was conducted. Heavy metal spiked soil into a

glass column was washed with 0.1% di-rhamnolipid biosurfactant solution. The results indicated that

di-rhamnolipid selectively removed heavy metals from soil in the order of Cd = Cr > Pb = Cu > Ni.

In turn, Das et al. [76] investigated the possibility of using the biosurfactant produced by marine

bacterium for removal of heavy metals from solutions. The positive role of marine biosurfactant in the

remediation of polyaromatic hydrocarbons was reported earlier [7], however there was no information

about the role of this biosurfactant in heavy metal remediation. The study revealed that tested anionic

biosurfactant was able to bind the metal ions and the percentage removal of Pb and Cd metals varied

with the different concentrations of metals and biosurfactants. The ability of biosurfactant of marine

origin to chelate toxic heavy metals and form an insoluble precipitate could be useful in treatment of

heavy metal containing wastewater.

Removal of heavy metals from sediments could be enhanced by use of solution containing

biosurfactant and inorganic compounds. For example, Dahrazma and Mulligan [77] reported the higher

rate of removal of copper and nickel from sediments by adding 1% NaOH to the solution of

rhamnolipid. Many metals mostly exist in the environment organic fraction, adding OH

-

to thesediment solubilizes this fraction, and thus, more metals are available for removal by a

rhamnolipid biosurfactant.

Another effective method for the remediation of heavy metals contaminated soil is biosurfactant

foam technology. Wang and Mulligan [78] evaluated the feasibility of using rhamnolipid foam to

remove Cd and Ni from a sandy soil. They reported that the use of foam had a significant effect on the

mobility of biosurfactant flowing in a porous medium and made a more uniform and efficient contact

of biosurfactant with the metals. Application of rhamnolipid foam increases efficiency and allows

removal of 73.2% and 68.1% of Cd and Ni, respectively, whereas the rhamnolipid solution flushed

only 61.7% and 51% of Cd and Ni, respectively. The system used for the experiment is presentedschematically by Wang and Mulligan [78].

The rate of heavy metal removal from soil strongly depends on its chemical composition. The

predominant constituent of the sand and silt fraction in many soils is quartz, thus quartz was chosen for

the bioremediation experiment. Ai et al. [68] studied recovery of the metal ions from quartz by

rhamnolipid. They observed that the best recovery efficiency from quartz, approximately 91.6% of the

sorbed Cd and 87.2% of the sorbed Zn, was achieved using 25 mM rhamnolipid concentration.

Biosurfactants were also used to evaluate their potential in arsenic mobilization from the mine

tailings [79]. The experimental results showed that introduction of rhamnolipid enhanced As

mobilization from the mine tailings significantly. The mobilization increased with the concentration of

biosurfactant and became relatively stable when the concentration of rhamnolipid was above

100 mgL1. It has been reported by Doong et al. [80] that the removal of heavy metals increased


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Int. J. Mol. Sci.2011, 12 647

linearly with increasing surfactant concentration below the CMC and remained relatively constant

above the CMC. The CMC of the biosurfactant used by Wang and Mulligan [79] was around

30 mgL1. The high concentration of rhamnolipid required in this experiment could be due to the

sorption of biosurfactant to the mine tailings and the dilution and binding effects of mine tailing

particles. The biosurfactant may be enhancing As mobilization by reducing the interfacial tension

between As and the mine tailings, by formation of aqueous complexes or micelles and by improving

the wettability of the mine tailings. The results from this research study indicated that biosurfactants

have potential to be used in the remediation of As-contaminated mine tailings and they can be also

effectively used to remove As from soils.

Besides the mobilization, biosurfactants can be involved in other processes connected with

remediation of heavy metals. They are used, for example, in entrapping of trivalent chromium in

micelles which provides bacterial tolerance and resistance towards high concentration of Cr(III).

Gnanamani et al. [81] studied the bioremediation of chromium (VI) by biosurfactant producing, marineisolate Bacillus sp. MTCC 5514. The remediation carried out by this strain proceeded via two

processes: reduction of Cr(VI) to Cr(III) by extracellular chromium reductase and entrapment of Cr(III)

by the biosurfactants. The first process transforms the toxic state of chromium into less-toxic state and

the second process prevents the bacterial cells from the exposure of chromium(III). Both reactions keep

bacterial cells active all the time and provide tolerance and resistance toward high hexavalent and

trivalent chromium concentrations.

4.2. Biosurfactants and Phytoremediation

Efficiency of phytoremediation of heavy metal contaminated soils can be increased by inoculation

of plants by biosurfactant-producing and heavy metal-resistant bacteria. Biosurfactant-producing

Bacillus sp. J119 strain was investigated for its capability to promote the plant growth and cadmium

uptake of rape, maize, sudangrass and tomato in soil contaminated with different levels of Cd [82]. The

study demonstrated that the tested strain could colonize the rhizosphere of all studied plants but its

application enhanced biomass and Cd uptake only in plant tissue of tomato. This means that root

colonization activity of the introduced strain is plant type influenced. However, further analyses of

interactions between the plants and biosurfactant-producing bacterial strain J119 may provide a new

microbe assisted-phytoremediation strategy for metal-polluted soils. Further work on the applicationsof biosurfactants and biosurfactants-producing bacteria in phytoremediation, especially in sites

co-contaminated with organic and metal pollutants is required.

5. Biosurfactants in Co-Contaminated Sites Remediation

It was estimated by the U.S. Environmental Protection Agency that 40% of sites are

co-contaminated with organics and metals pollutions [83]. The presence of toxic metals (lead,

cadmium, arsenic) in some cases causes inhibition of organic compound biodegradation [8385].

However, a review of the literature shows a number of possible approaches that can lower metal

bioavailability and/or increase microbial tolerance to metals. These include inoculation with

metal-resistant microorganisms, addition of materials like: clay mineralskaolinite and

montmorillonite, calcium carbonate, phosphate, chelating agents (EDTA), and bio- and


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Int. J. Mol. Sci.2011, 12 648

surfactants [83]. Biosurfactants produced by microorganisms show promise for enhancing organic

compound biodegradation in the presence of metals. Application of biosurfactants or microorganism

produced biosurfactants in in situ co-contaminated sites bioremediation seems to be more

environmentally compatible and more economical than using modified clay complexes or

metal chelators.

Sandrin et al. [84] showed that metal-complexing rhamnolipids reduced metal toxicity to allow

enhanced organic biodegradation by Burkholderia sp. under laboratory conditions. This research

demonstrated that rhamnolipids induced the release of lipopolisaccharide (LPS) from gram-negative

bacteria, Burkholderia sp., which does not produce rhamnolipid. The authors suggested that

rhamnolipid was able to reduce metal toxicity to microbial consortia in co-contaminated soils through a

combination of metal complexation and in the alteration of cell surface properties through the release

of lipopolisaccharide (LPS), resulting in enhanced bioremediation effect. Maslin and Maier [85]

studied the effect of rhamnolipids produced by various Pseudomonas aeruginosa strains on thephenanthrene degradation by indigenous populations in two soils co-contaminated with phenanthrene

and cadmium. The authors showed that rhamnolipids applied had the ability to complex cationic

metals, increasing the phenanthrene bioavailability [85]. The biodegradation of phenanthrene was

increased from 7.5 to 35% in one soil, and from 10 to 58% in the second soil, in response to

rhamnolipids application. As biosurfactants are degraded by soil populations in 23 weeks, Maslin and

Maier [85] used a pulsing strategy, in which new portions of rhamnolipids were added to the system to

maintain a constant level of biosurfactant during organic contaminant mineralization.

6. Conclusions and Future Perspectives

Application of biosurfactant and biosurfactant-producing bacteria in environmental technologies

(bioremediation and phytoremediation) has been studied. Both organic and inorganic contaminants can

be removed through different processes (physico-chemical and biological) in which biosurfactants are

involved. Due to their biodegradability and low toxicity, they are very promising for use in

environmental biotechnologies. The commercial success of biosurfactants is still limited by their high

production cost. Optimized growth conditions using cheap renewable substrates (agro-industrial

wastes) and novel, efficient methods for isolation and purification of biosurfactants could make their

production more economically feasible. Another important aspect regarding biological remediationtechnologies is the use of biosurfactant in the process on a large scale. To felicitate this process, new

techniques should be developed such as foams or micro-foams (colloidal gas aphrons-CGA) in

conjunction with biosurfactants.

Little is known about the potential of biosurfactant production by microorganisms in situ. Most of

the described studies were done under laboratory conditions. More efforts are required to evaluate the

biosurfactant production by microorganisms in situ and their role in biological remediation

technologies. Remediation systems with only one type of the contaminant have been studied to gain a

basic understanding. Only a few studies have also been completed on metal-organic pollutant

co-contaminated site remediation [86]. More information is required concerning the structures ofbiosurfactants, their interaction with soil and contaminants and scale up and cost effective biosurfactant

production [86]. For lowering the cost of biosurfactant production, commercially viable biological and


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Int. J. Mol. Sci.2011, 12 649

engineering solutions are required. One important point in this context is the use of low cost substrates

for production of biosurfactants.

A promising approach seems to be the application of inoculants of biosurfactant producing bacteria

in phytoremediation of hydrocarbon polluted soil to improve the efficiency of this technology.

Application of the biosurfactants in phytoremediation on a large scale requires studies to identify their

potential toxic effect on plants. Although the biosurfactants are thought to be ecofriendly, some

experiments indicated that under certain circumstances they can be toxic to the environment [87].

Nevertheless, careful and controlled use of these interesting surface active molecules will surely help in

the enhanced clean up of the toxic environmental pollutants and provide us with a clean environment.

Acknowledgements

This paper was prepared in connection with work done under the project No N N523 418237 from

the Polish Ministry of Science and Higher Education.

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